Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8(+) T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637), displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication.     

Adenovirus-vectored T cell vaccine for hepacivirus shows reduced effectiveness against a CD8 T cell escape variant in rats

There is an urgent need for a vaccine to prevent chronic infection by hepatitis C virus (HCV) and its many genetic variants. The first human vaccine trial, using recombinant viral vectors that stimulate pan-genotypic T cell responses against HCV non-structural proteins, failed to demonstrate efficacy despite significant preclinical promise. Understanding the factors that govern HCV T cell vaccine success is necessary for design of improved immunization strategies. Using a rat model of chronic rodent hepacivirus (RHV) infection, we assessed the impact of antigenic variation and immune escape upon success of a conceptually analogous RHV T cell vaccine. Naïve Lewis rats were vaccinated with a recombinant human adenovirus expressing RHV non-structural proteins (NS)3-5B and later challenged with a viral variant containing immune escape mutations within major histocompatibility complex (MHC) class I-restricted epitopes (escape virus). Whereas 7 of 11 (64%) rats cleared infection caused by wild-type RHV, only 3 of 12 (25%) were protected against heterologous challenge with escape virus. Uncontrolled replication of escape virus was associated with durable CD8 T cell responses targeting escaped epitopes alone. In contrast, clearance of escape virus correlated with CD4 T cell helper immunity and maintenance of CD8 T cell responses against intact viral epitopes. Interestingly, clearance of wild-type RHV infection after vaccination conferred enhanced protection against secondary challenge with escape virus. These results demonstrate that the efficacy of an RHV T cell vaccine is reduced when challenge virus contains escape mutations within MHC class I-restricted epitopes and that failure to sustain CD8 T cell responses against intact epitopes likely underlies immune failure in this setting. Further investigation of the immune responses that yield protection against diverse RHV challenges in this model may facilitate design of broadly effective HCV vaccines.     

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.     

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.     

Sequence Variation in the Gene Encoding the Nonstructural 3 Protein of Hepatitis C Virus: Evidence for Immune Selection

To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3) gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star topology suggesting that a wild-type HCV sequence is maintained, unlike HIV. Received: 2 November 2000 / Accepted: 1 October 2001     

Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.     

Minimal T-cell-stimulatory sequences and spectrum of HLA restriction of immunodominant CD4+ T-cell epitopes within hepatitis C virus NS3 and NS4 proteins

The hepatitis C virus (HCV)-specific CD4+ T-cell response against nonstructural proteins is strongly associated with successful viral clearance during acute hepatitis C. To further develop these observations into peptide-based vaccines and clinical immunomonitoring tools like HLA class II tetramers, a detailed characterization of immunodominant CD4+ T-cell epitopes is required. We studied peripheral blood mononuclear cells from 20 patients with acute hepatitis C using 83 overlapping 20-mer peptides covering the NS3 helicase and NS4. Eight peptides were recognized by > or = 40% of patients, and specific CD4+ T-cell clones were obtained for seven of these and three additional, subdominant epitopes. Mapping of minimal stimulatory sequences defined epitopes of 8 to 13 amino acids in length, but optimal T-cell stimulation was observed with 10- to 15-mers. While some epitopes were presented by different HLA molecules, others were presented by only a single HLA class II molecule, which has implications for patient selection in clinical trials of peptide-based immunotherapies. In conclusion, using two different approaches we identified and characterized a set of CD4+ T-cell epitopes in the HCV NS3-NS4 region which are immunodominant in patients achieving transient or persistent viral control. This information allows the construction of a valuable panel of HCV-specific HLA class II tetramers for further study of CD4+ T-cell responses in chronic hepatitis C. The finding of immunodominant epitopes with very constrained HLA restriction has implications for patient selection in clinical trials of peptide-based immunotherapies.     

Anti-Hepatitis C Virus T-Cell Immunity in the Context of Multiple Exposures to the Virus

Characterisation of Hepatitis C virus (HCV)-specific CD8⁺ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8⁺ T-cell targets. Peptides representing these putative HCV CD8⁺ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design.     

Virus-specific T-cell immunity correlates with control of GB virus B infection in marmosets

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-gamma ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.     

Escape mutations alter proteasome processing of major histocompatibility complex class I-restricted epitopes in persistent hepatitis C virus infection

Mutations in hepatitis C virus (HCV) genomes facilitate escape from virus-specific CD8+ T lymphocytes in persistently infected chimpanzees. Our previous studies demonstrated that many of the amino acid substitutions in HCV epitopes prevented T-cell receptor recognition or binding to class I major histocompatibility complex molecules. Here we report that mutations within HCV epitopes also cause their destruction by changing the pattern of proteasome digestion. This mechanism of immune evasion provides further evidence of the potency of CD8+ T-cell selection pressure against HCV and should be considered when evaluating the significance of mutations in viral genomes from persistently infected chimpanzees and humans.     

Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.     

Mutations in immunodominant T cell epitopes derived from the nonstructural 3 protein of hepatitis C virus have the potential for generating escape variants that may have important consequences for T cell recognition

One of the most disturbing features of hepatitis C virus (HCV) is its long-term persistence in the host. One hypothesis to explain this phenomenon is that HCV escapes immune recognition through its intrinsic hypermutability. To determine whether immunodominant T cell epitopes derived from HCV nonstructural 3 (NS3) protein might be subject to sequence variations leading to escape mutants, we examined sequence variations of one IL-2-producing epitope, NS3358-375, and one IL-10-producing epitope, NS3505-521. By PCR amplification, cloning, and sequencing, we observed significant sequence variations in the two epitopes, although the selection intensity for each epitope was different. For NS3358-375, more variants were observed, and for NS3505-521, fewer mutations were observed. Moreover, functional studies revealed that three NS3358-375 and one NS3505-521 variants failed to stimulate T cell proliferation, and two other NS3358-375 and NS3505-521 variants weakly stimulated T cell responses. Our results are consistent with immune selection of viral variants at the epitope level, which may enable HCV to evade host defenses over time.     

Effect of bottlenecking on evolution of the nonstructural protein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection

Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 x 10(-3) mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.     

Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.     

Compensatory mutations restore the replication defects caused by cytotoxic T lymphocyte escape mutations in hepatitis C virus polymerase

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.     

Molecular and immunological significance of chimpanzee major histocompatibility complex haplotypes for hepatitis C virus immune response and vaccination studies

The chimpanzee is a critical animal model for studying cellular immune responses to infectious pathogens such as hepatitis B and C viruses, human immunodeficiency virus, and malaria. Several candidate vaccines and immunotherapies for these infections aim at the induction or enhancement of cellular immune responses against viral epitopes presented by common human major histocompatibility complex (MHC) alleles. To identify and characterize chimpanzee MHC class I molecules that are functionally related to human alleles, we sequenced 18 different Pan troglodytes (Patr) alleles of 14 chimpanzees, 2 of them previously unknown and 3 with only partially reported sequences. Comparative analysis of Patr binding pockets and binding assays with biotinylated peptides demonstrated a molecular homology between the binding grooves of individual Patr alleles and the common human alleles HLA-A1, -A2, -A3, and -B7. Using cytotoxic T cells isolated from the blood of hepatitis C virus (HCV)-infected chimpanzees, we then mapped the Patr restriction of these HCV peptides and demonstrated functional homology between the Patr-HLA orthologues in cytotoxicity and gamma interferon (IFN-gamma) release assays. Based on these results, 21 HCV epitopes were selected to characterize the chimpanzees' cellular immune response to HCV. In each case, IFN-gamma-producing T cells were detectable in the blood after but not prior to HCV infection and were specifically targeted against those HCV peptides predicted by Patr-HLA homology. This study demonstrates a close functional homology between individual Patr and HLA alleles and shows that HCV infection generates HCV peptides that are recognized by both chimpanzees and humans with Patr and HLA orthologues. These results are relevant for the design and evaluation of vaccines in chimpanzees that can now be selected according to the most frequent human MHC haplotypes.     

Differences in hepatitis C virus (HCV)-specific CD8 T-cell phenotype during pegylated alpha interferon and ribavirin treatment are related to response to antiviral therapy in patients chronically infected with HCV

CD8 T cells play a major role in antiviral immune responses. Their importance for progression to chronic hepatitis C and response to treatment are still unclear. To address these issues, hepatitis C virus (HCV)-specific CD8 T-cell responses were monitored, at the single-cell level, using HLA class I pentamers specific for HCV core and HCV NS3 epitopes, in 23 chronically infected patients during treatment with pegylated alpha interferon and ribavirin. Patients who presented a sustained-response to therapy had stronger HCV-specific CD8 T-cell responses at all time points studied. Moreover, there were clear differences in the phenotypes of these cells during therapy: in responder patients, terminally differentiated effector cells increased more rapidly, and their frequency was always higher than in nonresponder patients. Sustained-responder patients also showed a higher frequency of HCV-specific CD8 T cells producing cytotoxic factors. Overall, a late and inefficient differentiation process of HCV-specific CD8 T cells might be associated with lack of response to treatment. A better knowledge of the mechanisms underlying this impairment may be important for the development of new therapeutic strategies to maintain, restore, or increase CD8 T-cell effectiveness in chronic HCV infection.     

Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.     

Viral adaptation to host immune responses occurs in chronic hepatitis B virus (HBV) infection, and adaptation is greatest in HBV e antigen-negative disease

Hepatitis B virus (HBV)-specific T-cell responses are important in the natural history of HBV infection. The number of known HBV-specific T-cell epitopes is limited, and it is not clear whether viral evolution occurs in chronic HBV infection. We aimed to identify novel HBV T-cell epitopes by examining the relationship between HBV sequence variation and the human leukocyte antigen (HLA) type in a large prospective clinic-based cohort of Asian patients with chronic HBV infection recruited in Australia and China (n = 119). High-resolution 4-digit HLA class I and II typing and full-length HBV sequencing were undertaken for treatment-naïve individuals (52% with genotype B, 48% with genotype C, 63% HBV e antigen [HBeAg] positive). Statistically significant associations between HLA types and HBV sequence variation were identified (n = 49) at 41 sites in the HBV genome. Using prediction programs, we determined scores for binding between peptides containing these polymorphisms and associated HLA types. Among the regions that could be tested, HLA binding was predicted for 14/18 (78%). We identified several HLA-associated polymorphisms involving likely known anchor residues that resulted in altered predicted binding scores. Some HLA-associated polymorphisms fell within known T-cell epitopes with matching HLA restriction. Enhanced viral adaptation (defined as the presence of the relevant HLA and the escaped amino acid) was independently associated with HBeAg-negative disease (P = 0.003). Thus, HBV appears to be under immune pressure in chronic HBV infection, particularly in HBeAg-negative disease.     

Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus

Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demonstrated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCV morphogenesis is poorly understood. We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 orders of magnitude higher than that of wild-type virus. Sequence analysis identified a total of 16 amino acid mutations in core (C), E1, NS2, NS3, NS5A, and NS5B, with the majority of mutations clustered in NS5A. Reverse genetic analysis of these mutations individually or in different combinations demonstrated that amino acid mutations in NS2 and NS5A markedly enhanced HCV production. Additionally, mutations in C, E1, NS3, and NS5B synergistically promoted HCV production in the background of NS2 and NS5A mutations. Adaptive mutations in NS5A domains I, II, and III independently enhanced HCV production, suggesting that all three domains of NS5A are important for HCV morphogenesis. More importantly, adaptive mutations greatly enhanced physical interactions among HCV structural and NS proteins, as determined by studies with coimmunoprecipitation and mammalian two-hybrid assays. Collectively, these findings demonstrate that adaptive mutations can enhance specific protein-protein interactions among viral structural and NS proteins and therefore promote the assembly of infectious HCV particles.